The major histocompatability complex (MHC) on Chromosome 6 is the most variable and gene-dense region of the human genome. The genes in the MHC can be associated with all known autoimmune disorders and play a critical role in transplant rejection. Unfortunately, the molecular details of the MHC have been exceedingly difficult to study due to the inherent difficulty in sequencing this region of the genome. Specifically, the large size (~4 Mb) and extreme variability of the MHC greatly complicate conventional sequencing methods. Next-generation DNA sequencers, known as "massively-parallel" sequencers, have the ability to sequence multiple megabases of highly-variable sequence in a matter of hours. These instruments are designed for high-throughput "shotgun" sequencing of whole genomes, however, and cannot presently be used to sequence discreet genomic loci such as the MHC. This proposal concerns the development of engineered, MHC-specific restriction enzymes called "meganucleases" for use in MHC sequencing. We propose that engineered meganucleases can be produced which specifically recognize and cut long, unique DNA sites found in or near the MHC. One such meganuclease has already been produced and tested at Precision BioSciences. We propose to develop 4 additional MHC-specific meganucleases and to use these to cut the MHC locus out of purified human chromosomal DNA for subsequent sequencing on a massively-parallel instrument. If this project is successful, it will be possible to cost-effectively sequence the entire MHC locus from a patient's blood sample in a matter of days. The impacts that such a technology would have on transplantation success and clinical and research immunology would be enormous. PUBLIC HEALTH RELEVANCE: The genes in the MHC region of the human genome play important roles in autoimmune diseases (such as type 1 diabetes and rheumatoid arthritis), inflammatory diseases (such as Crohn's disease), and transplant rejection. Precision BioSciences is proposing to develop a technology that will help to quickly and cost-effectively determine the DNA sequence of the MHC region from a patient's blood sample. This will greatly assist in the study and diagnosis of a wide range of diseases and will improve transplant success.